Stromal cell-derived factor-1 overexpression induces
gastric dysplasia through expansion of stromal
myofibroblasts and epithelial progenitors
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Citation
Shibata, W. et al. “Stromal Cell-derived Factor-1 Overexpression
Induces Gastric Dysplasia Through Expansion of Stromal
Myofibroblasts and Epithelial Progenitors.” Gut (2012). Copyright
© 2012 BMJ Publishing Group Ltd & British Society of
Gastroenterology
As Published
http://dx.doi.org/10.1136/gutjnl-2011-301824
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Thu May 26 20:16:45 EDT 2016
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http://hdl.handle.net/1721.1/72115
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Gut Online First, published on February 23, 2012 as 10.1136/gutjnl-2011-301824
Gastric cancer
ORIGINAL ARTICLE
Stromal cell-derived factor-1 overexpression induces
gastric dysplasia through expansion of stromal
myofibroblasts and epithelial progenitors
Wataru Shibata,1 Hiroshi Ariyama,1 Christoph Benedikt Westphalen,1
Daniel L Worthley,1 Sureshkumar Muthupalani,2 Samuel Asfaha,1
Zinaida Dubeykovskaya,1 Michael Quante,3 James G Fox,2 Timothy C Wang1
< Additional materials are
published online only. To view
these files please visit the
journal online (http://gut.bmj.
com/content/early/recent).
1
Department of Medicine,
Division of Digestive and Liver
Disease, College of Physicians
and Surgeons, Columbia
University, New York, New
York, USA
2
Division of Comparative
Medicine, Massachusetts
Institute of Technology,
Cambridge, Massachusetts,
USA
3
II. Medizinische Klinik, Klinikum
rechts der Isar, Technische
Universität München, Munich,
Germany
Correspondence to
Dr Professor Timothy C Wang,
Chief, Division of Digestive and
Liver Diseases, Silberberg
Professor of Medicine,
Department of Medicine and
Irving Cancer Research Center,
Columbia University Medical
Center, 1130 Street Nicholas
Avenue, Room #925, New
York, NY 10032-3802, USA;
tcw21@columbia.edu
Revised 4 January 2012
Accepted 11 January 2012
ABSTRACT
Objective Stromal cell-derived factor-1 (SDF-1/CXCL12),
the main ligand for CXCR4, is overexpressed in human
cancer. This study addressed the precise contribution of
SDF-1 to gastric carcinogenesis.
Design SDF-1 transgenic mice were created and
a Helicobacter-induced gastric cancer model was used in
combination with H/K-ATPase-IL-1b mice. Gastric tissue
was analysed by histopathology and cells isolated from
the stomach were analysed by molecular biological
methods.
Results Analysis of the H/K-ATPase/SDF-1 transgenic
(SDF-Tg) mice showed that SDF-1 overexpression results
in significant gastric epithelial hyperproliferation, mucous
neck cell hyperplasia and spontaneous gastric dysplasia
(wild-type mice 0/15 (0%) vs SDF-Tg mice 4/14 (28.6%),
p¼0.042, Fisher exact test) but has minimal effects on
inflammation. SDF-Tg mice also showed a dramatic
expansion of a-smooth muscle actin-positive
myofibroblasts and CXCR4-expressing gastric epithelial
cells in the progenitor zone, both of which preceded the
development of significant gastritis or dysplasia. Gremlin
1-expressing mesenchymal stem cells, the putative
precursors of myofibroblasts, were also increased within
the dysplastic stomachs of SDF-Tg mice and showed
chemotaxis in response to SDF-1 stimulation. SDF-1
overexpression alone resulted in minimal recruitment of
haematopoietic cells to the gastric mucosa, although
macrophages were increased late in the disease. When
SDF-Tg mice were crossed with H/K-ATPase-IL-1b mice
or infected with Helicobacter felis, however, there were
dramatic synergistic effects on recruitment of bone
marrow-derived cells and progression to preneoplasia.
Conclusion Activation of the SDF-1/CXCR4 axis can
contribute to early stages of carcinogenesis primarily
through recruitment of stromal cells and modulation of
the progenitor niche.
Significance of this study
What is already known on this subject?
< Stromal cell-derived factor-1 (SDF-1) and its
cognate receptor CXCR4 mediate many biological functions such as embryonic development,
organ homeostasis, angiogenesis and immune
system modulation.
< Studies in murine cancer models have
shown that SDF-1 produced by myofibroblasts
recruits endothelial cells and contributes to
tumorigenesis.
< SDF-1 mRNA and serum SDF-1 are upregulated
in both mice and human gastric cancer and
blocking SDF/CXCR4 reduces tumour growth
and the development of ascites.
What are the new findings?
< Overexpression of SDF-1 in the stomach caused
a significant increase in gastric epithelial cell
proliferation, induced inflammation and
promoted gastric tumour development, particularly in combination with inflammatory stimuli
such as Helicobacter infection or interleukin
1b (IL-1b).
< SDF-1 recruited F4/80-positive macrophages
and CD11b-positive myeloid cells and induced
gastric epithelial proliferation partly through the
activation of SDF/CXCR4 signalling and its
downstream Erk/PI3kinase.
< SDF-1 overexpression recruited CXCR4-positive
mesenchymal stem cells and the expansion
of myofibroblasts in the gastric stem cell
niche. This was associated with an increase in
K19-positive epithelial cells.
< SDF-1 promoted gastric epithelial proliferation
partly through CXCR4-positive gastric tissue
stem/progenitor cells.
INTRODUCTION
Gastric cancer is the second leading cause of cancer
worldwide, and it is now well established that
Helicobacter pylori-associated chronic inflammation
plays a pivotal role in triggering the sequence from
chronic gastritis to cancer.1 The active stromal
remodelling that accompanies chronic inflammation is critical to the initiation and progression of
cancer.2 Thus, there has been increasing interest to
identify the key chemokines and cytokines that
mobilise inflammatory and mesenchymal cells,
leading to cancer.3
The chemokine stromal cell-derived factor-1
(SDF-1), also known as CXCL12, is a constitutively
expressed and inducible chemokine that plays
a fundamental role in embryonic development,
organ homeostasis, angiogenesis and immune
system modulation. SDF-1 binds to and initiates
Shibata W, Ariyama
H, Westphalen
CB, et
al. Gutemployer)
(2012). doi:10.1136/gutjnl-2011-301824
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Article
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their
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Gastric cancer
MSCs and CAFs, constituting the functional mesenchymal
niche from the BM to sites of chronic injury. This recruited
mesenchymal niche is likely to promote local stromal remodelling as well as cancer development.10 19 24 Nevertheless, the full
contribution of SDF-1 in gastric carcinogenesis is still unknown.
In order to better define the precise role of SDF-1 in gastric
carcinogenesis, we generated mice overexpressing SDF-1 in
gastric parietal cells and examined its relevance to epithelial and
stromal events in cancer.
Significance of this study
How might it impact on clinical practice in the foreseeable
future?
< SDF-1 drives cancer through cytokine-related recruitment of
macrophages and chemotaxis of bone marrow-derived
mesenchymal stem cells/myofibroblasts.
< In addition, the SDF-1/CXCR4 pathway plays a key role in the
development of gastric carcinogenesis through direct modulation of CXCR4-positive stem/progenitor epithelial cells. SDF/
CXCR4 signalling is an attractive target for the prevention and
treatment of gastric cancer.
MATERIALS AND METHODS
signalling through two G-protein-coupled receptors, CXCR4 and
CXCR7. In the bone marrow (BM), SDF-1 plays a critical role in
the initial localisation, retention and support of CXCR4
haematopoietic stem cells (HSCs).4 SDF-1 is highly expressed in
BM stromal cells and contributes to the BM niche. Gene
knockout of either SDF-1 or CXCR4 results in impaired
haematopoiesis and embryonic lethality,5e7 and inactivation of
the SDF/CXCR4 signalling pathway promotes the mobilisation
of HSCs into peripheral blood8 or mobilisation of neutrophils.9
Taken together, there is strong evidence that SDF-1/CXCR4
interactions modulate the engraftment, retention and release of
haematopoietic cells from the BM.
It is not known whether SDF-1 also promotes the mobilisation and recruitment of haematopoietic cells into peripheral
tissues. Indeed, SDF-1 is often upregulated in damaged tissues as
a result of hypoxia or cellular apoptosis and increased SDF-1
plasma levels correlate with mobilisation of proangiogenic BM
cells.10 The cognate receptor for SDF-1, CXCR4, is expressed on
most BM-derived haematopoietic cells, including HSCs, endothelial precursor cells, immature myeloid cells, macrophages and
lymphocytes11 and, through activation of phosphoinositide 3kinase, can regulate chemotaxis. While release of local SDF-1,
such as following ischaemia, can mobilise haematopoietic cells,12
the recruitment of inflammatory cells is usually limited in
the absence of injury, suggesting that SDF-1 is not sufficient for
BM-derived cell recruitment.13 14
SDF-1 is one of the major chemokines consistently overexpressed in most solid tumours,15 16 where it contributes to
carcinogenesis as an autocrine growth factor as well as
promoting angiogenesis and the recruitment of BM cells to the
tumour microenvironment.17e19 While the CXCR4 receptor is
primarily localised to the cancer cells,11 20 the major source of
the SDF-1 ligand in solid tumours appears to be stromal cells,
particularly cancer-associated fibroblasts (CAFs).17 CAFs exhibit
properties of myofibroblasts, including expression of a-smooth
muscle actin (aSMA), and promote the growth of tumours
through their ability to secrete SDF-1.17 Recent studies indicate
that BM-derived mesenchymal stem cells (MSCs) represent one
potential source for CAFs, and MSCs recruited into the cancer
microenvironment are able to differentiate into CAF-like
myofibroblasts.21e23 SDF-1 secreted by CAFs is important for
both the migration and survival of MSCs in vitro.19 The overexpression of interleukin 1b (IL-1b) as well as chronic gastric
Helicobacter infection both increase SDF-1 expression in the
gastric mucosa. Furthermore, the recruitment of MSCs and
CAFs, mediated by both SDF-1 and transforming growth factor
(TGF) b, resulted in hyperproliferation of gastric epithelial
cells.19 Thus, SDF-1 released by CAFs potentially recruits both
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All animal studies were approved by the Institutional Animal
Care and Use Committee at Columbia University. SDF transgenic mice were generated as described in the online supplement.
CXCR4-EGFP mice were provided by Richard J Miller (Northwestern University Medical School, Chicago, Illinois, USA).
Details of the mice used in this study, all protocols for bacterial
culture, chronic H felis infection model, histological evaluation,
immunohistochemical studies, ELISA, real-time qRT-PCR assay
of H felis infection in mouse stomachs, proinflammatory CC
chemokines and cytokines are given in the online supplement.
RESULTS
Gastric overexpression of SDF-1 results in development of
spontaneous gastric cancer
We generated H/K-ATPase/hSDF-1a transgenic mice (SDF-Tg)
that expressed murine SDF-1a specifically in gastric parietal cells
(supplementary figure 1A). Two independent lines (lines 3 and 6)
of SDF-Tg mice were identified by ELISA of gastric mucosa for
SDF-1 or v5 tag (supplementary figure 1BeE). We confirmed
that the murine Met-SDF-1V5x6His protein derived from the
construct was biologically active and could induce lymphocyte
chemotaxis in a dose-dependent manner, and the tagged SDF-1
protein bound surface proteoglycans with only slightly less
efficiency than commercial recombinant SDF-1 (PeproTech Inc.,
Rocky Hill, NJ, USA.) lacking initial Met (data not shown). We
backcrossed SDF-Tg mice to C57B6/J mice at least six times
prior to further studies.
SDF-Tg mice exhibited markedly elevated expression of SDF-1
mRNA in the gastric mucosa (supplementary figure 1E), which
was greater than the expression level observed in the BM.
Interestingly, SDF-Tg mice aged between 3 and 12 months
showed minimal inflammation in the stomach but nevertheless
exhibited gastric hyperplasia and metaplasia. Young SDF-Tg
mice (2e6 months) showed only a slight increase in the number
of inflammatory cells determined by immunohistochemistry
and FACS (supplementary figure 2A,B). By 18 months of age,
however, the SDF-Tg mice exhibited dysplasia with marked
cystic dilation of glands in corpus and antral tumours (figure 1).
Furthermore, we observed a significant increase in chronic
inflammatory cells at these later time points. While not evident
in younger mice (not shown), there was a significant increase in
the number of F4/80-positive cells and myeloperoxidase activity
in older SDF-Tg mice (>12 months) compared with wild-type
(WT) mice (supplementary figure 2C).
These results suggest that SDF-1 is able to induce gastric
neoplasia despite minimal early effects on the recruitment of
chronic inflammatory cells. In particular, the findings implied
that, while SDF-1 is clearly able to recruit and retain CXCR4positive haematopoietic cells within the BM,25 overexpression of
SDF-1 in the stomach alone is not sufficient to induce the
recruitment of haematopoietic cells to the stomach. We further
tested this concept by transplantation of SDF-Tg and WT mice
Shibata W, Ariyama H, Westphalen CB, et al. Gut (2012). doi:10.1136/gutjnl-2011-301824
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Gastric cancer
Figure 1 SDF-Tg mice developed
spontaneous gastric dysplasia.
Representative macroscopic and
histological micrographs from SDF-Tg
mice at 18 months (original
magnifications 3100).
with GFP-labelled BM and followed the mice for up to 1 year.
SDF-Tg transgenic mice showed a slight increase in GFP-labelled
haematopoietic cells compared with WT mice at 1 year (data
not shown), indicating that overexpression of SDF-1 alone has
a weak and/or indirect effect on the recruitment of haematopoietic cells to the stomach. Nevertheless, the findings suggest
that SDF-1 is able to drive epithelial proliferation independent of
effects on haematopoietic cells. There were no histological
alterations in other organs examined including intestine, liver,
lung, and kidney in SDF-Tg mice (data not shown).
SDF-1 overexpression accelerates inflammation-induced gastric
tumorigenesis
Given the absence of robust inflammation and the overall low
incidence of gastric neoplastic lesions in SDF-Tg mice, we
postulated that the induction of chronic inflammation in these
animals might further accelerate the development of neoplasia.
Thus, we employed H felis infection in SDF-Tg mice in order to
analyse the impact of SDF-1 overexpression in an established
model of inflammation-associated gastric carcinogenesis.26 The
mice were all of the same mixed genetic background (C57BL/63
CBA), and examined at time points up to 18 months after H felis
infection (MPI). Every histopathological parameter tended to be
more severe in H felis-infected SDF-Tg mice compared with
H felis-infected WT mice up to 12 MPI. At time points between
15 and 18 MPI there were even more prominent histopathological changes, with significantly greater degrees of pseudopyloric metaplasia, oxyntic atrophy and foveolar hyperplasia in
H felis-infected SDF-Tg (figure 2A,B). Interestingly, only the
H felis-infected SDF-Tg mice exhibited gastric dysplasia and
mucous metaplasia at 15e18 MPI, while no H felis-infected WT
mice developed severe dysplasia. Additionally, SDF-Tg mice
infected with H felis showed increased gastric expression of
IL-1b, heparin-binding epidermal growth factor and amphiregulin (figure 2C) and increased serum levels of IL-6 compared
with WT mice infected with H felis (figure 2D).
The findings regarding IL-1b were particularly interesting,
given previous studies in humans that had pointed to an
important role for the IL-1b gene locus in gastric cancer susceptibility.27 Since gastric specific overexpression of IL-1b in transgenic mice was shown to be sufficient for the recruitment of
myeloid cells to the stomach and the induction of gastric
cancer,18 we crossed H/K-ATPase-IL-1b mice with SDF-Tg mice
in order to determine the impact on carcinogenesis. At very early
time points (eg, 1e1.5 months) we found that SDF-1/IL-1b
double transgenic mice showed significantly increased inflammatory cell infiltration, severe gastric atrophy and intestinal
metaplasia in stomach compared with IL-1b single transgenic
mice (supplementary figure 3A,B). The levels of IL-6 in gastric
tissue were also significantly higher in SDF-1/IL-1b double
transgenic mice compared with IL-1b single transgenic mice
(supplementary figure 3C). In combination with either H felis
infection or IL-1b overexpression, the number of F4/80 and
CD11b-positive cells was significantly increased in SDF-Tg mice
compared with their respective controls (figure 3A,B). These data
suggest that, although SDF-1 alone is not sufficient to strongly
promote chronic gastritis, it synergises with Helicobacter infection
or IL-1b overexpression in the recruitment of myeloid cells and
the induction of atrophic gastritis and gastric preneoplasia.
SDF-1 extends survival and promotes the function of myeloid
cells in vitro
Given that SDF-1 synergised with IL-1b in the induction of
gastritis, we wondered whether SDF-1 overexpression could
increase the abundance of macrophages and the development of
dysplasia through an effect on macrophage survival, as previously suggested.7 We isolated primary peritoneal macrophages
using 4% thioglycolate and then stimulated the macrophages
with either recombinant SDF-1 (rSDF-1) or gastric extract from
SDF-Tg mice or WT mice. After 48 h of serum-free incubation,
primary macrophages showed a significant increase in survival
after administration with either rSDF-1 or gastric extract from
SDF-Tg mice (supplementary figure 4A). We also analysed the
effect of SDF-1 on cytokine production in primary macrophages.
There was no IL-6 production after stimulation with rSDF-1
alone; however, rSDF-1 administration showed a significant
synergistic effect on the production of IL-6 from primary
macrophages co-stimulated with lipopolysaccharide (supplementary figure 4B). We found that the increase in cytokine
production was also associated with NF-kB signalling, confirmed
by experiments using NF-kB inhibitor, MG132 (data not
shown). Taken together, these results suggest that, apart from
Shibata W, Ariyama H, Westphalen CB, et al. Gut (2012). doi:10.1136/gutjnl-2011-301824
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Figure 2 Overexpression of stromal cell-derived factor-1 (SDF-1) accelerates the development of gastric inflammation and dysplasia in the setting of
Helicobacter felis infection. (A) Representative stomach sections. (B) Pathological scores from SDF-Tg mice and WT mice infected with H felis for
15e18 months (n¼5 per group). (C) qRT-PCR of gastric mucosa for the indicated genes. Gene expression was normalised to 104 copies of GAPDH
levels and the expression of each gene relative to WT mice is shown (n¼5 per group). (D) Serum interleukin 6 (IL-6) levels from WT and SDF-Tg mice
after 15 MPI were measured by ELISA (n¼5 per group). Data shown are mean6SE. *p<0.05.
its role as a weak chemoattractant for myeloid cells, SDF-1
directly contributes to the survival and function of myeloid cells.
SDF-1 induces epithelial proliferation and hyperplasia in part
through CXCR4
SDF-1 overexpression increases gastric epithelial proliferation.
Immunostaining for Ki67 was increased in both the corpus and
antrum of SDF-Tg mice compared with WT mice, irrespective of
H felis infection (figure 4A and supplementary figure 5A).
Compared with WT mice, the increase in gastric proliferation in
SDF-Tg mice led over time to gastric hyperplasia, particularly in
the gastric pit and mucous neck regions. The number of K19positive cells, previously suggested to be gastric pit and
progenitor cells,28 was significantly increased in SDF-Tg mice
compared with WT mice (supplementary figure 5B). The
number of parietal cells, however, was not significantly different
in SDF-Tg mice and WT mice, suggesting that overexpression of
SDF-1 somehow promoted the differentiation of the gastric
epithelial stem cells towards the K19-expressing pit cell lineage,
but not the glandular lineage.29
In order to confirm that the CXCR4 receptor mediated the
proliferative response to SDF-1, we administered the CXCR4
antagonist AMD3100 to a cohort of SDF-Tg mice and WT
controls. AMD3100 was administered to mice for 2 weeks. The
mice were then killed and gastric epithelial proliferation assessed
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by Ki-67 immunostaining. AMD3100 significantly reduced the
gastric epithelial proliferation index of the SDF-Tg mice to a level
approaching that of untreated WT mice (figure 4B), consistent
with a CXCR4-dependent pathway. Moreover, phosphorylatedAkt (p-Akt) and -Erk (p-Erk) could be detected in the bottom
third of the antral glands where CXCR4-expressing cells reside,
and p-Akt and p-Erk positive cells were significantly increased
both in the antrum and the corpus of SDF-Tg mice (figure 4C and
supplementary figure 6). The increase in p-Akt or p-Erk was
abrogated by the administration of AMD3100 (figure 4C and
supplementary figure 6). Activation of CXCR4 by SDF-1a has
previously been shown to result in the activation of ERK and
PI3K/Akt pathways, and downstream effectors of these pathways are reported to include IL-6.30 Taken together, these data
suggest that SDF-1a stimulates gastric epithelial proliferation
through Erk and PI3K/Akt-dependent mechanisms.
We have recently shown that the trefoil factor 2 (TFF2)
functions as a partial antagonist to the CXCR4 receptor, with
inhibitory effects on chemotaxis in response to SDF-1 through
competitive inhibition of CXCR4.31 We therefore crossed SDFTg mice with TFF2 null mice to test whether endogenous TFF2
might dampen the SDF-1-dependent proliferation and inflammation. SDF/TFF2-/- mice at 12 months of age showed
increased proliferation and a dramatic increase in histopathological scores for gastric inflammation, oxyntic atrophy,
Shibata W, Ariyama H, Westphalen CB, et al. Gut (2012). doi:10.1136/gutjnl-2011-301824
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Gastric cancer
Figure 3 SDF-Tg mice showed
increase in F4/80- and CD11b-positive
macrophages in stomach with
Helicobacter felis infection or interleukin
1b (IL-1b) overexpression. (A) Number
of F4/80-positive cells per high power
fields in WT and SDF-Tg mice
determined by counting positive cells
per high power fields (n¼5 per group,
15-month-old mice). (B) F4/80- or
CD11b-positive cells were increased in
SDF/IL-1b-Tg mice; representative
micrographs from stained paraffin
sections from IL-1b and SDF/IL-1b mice
(n¼5 per group). Original magnification
3100. Data shown are mean6SE.
*p<0.05 in each comparison indicated.
hyperplasia and metaplasia compared with SDF-Tg alone
(supplementary figure 7A,B). In addition, we noted that SDF-1/
TFF2-/- mice showed markedly increased epithelial proliferation
compared with age-matched SDF-1 transgenic mice as assessed
by Ki67 immunohistochemistry (supplementary figure 7C,D).
These results suggest that the development of gastric preneoplasia in SDF-Tg mice involves activation of the SDF/CXCR4
signalling pathway.
To determine whether overexpression of SDF-1 directly
induced gastric epithelial proliferation, we identified potential
SDF-1 target cells expressing the CXCR4 receptor in the
stomach mucosa using CXCR4-EGFP BAC transgenic mice
(supplementary figure 8A).32 In the corpus these CXCR4-EGFP
(+) cells were limited to the gastric isthmus, the known
progenitor zone of the oxyntic mucosa. Furthermore, CXCR4EGFP (+) cells were increased when CXCR4-EGFP mice were
crossed with SDF-Tg mice compared with WT-CXCR4-EGFP
mice (supplementary figure 8B). In the gastric antrum, however,
the bottom third of the glands showed strong GFP-positive
signals that appeared to overlap with the known location of
antral stem and progenitor cells (supplementary figure 8C).33
Taken together, these data are consistent with a model in which
overexpression of SDF-1 in the stomach could promote gastric
epithelial cell proliferation directly, at least in part, through
activation of CXCR4-expressing gastric progenitor cells.
SDF-1 induces expansion of stromal myofibroblasts through
recruitment of CXCR4-expressing MSCs
While there were some gastric epithelial cells in the corpus that
were positive for CXCR4-EGFP, there were clearly multiple
stromal cells in the region of the gastric progenitor zone
(isthmus) that were also positive for CXCR4-EGFP (supplementary figure 8A). Many of these had the appearance of
stromal fibroblasts or myofibroblasts. Recent studies have
suggested that aSMA-positive myofibroblasts contribute to the
gastric stem cell niche,34 and that expansion of these cells
contributes to the development of gastric neoplasia.19 Consequently, we examined the effects of SDF-1 overexpression on of
aSMA-positive myofibroblasts by crossing SDF-Tg mice with
aSMA-RFP (red fluorescent protein) reporter mice19 to generate
SDF-Tg/aSMA-RFP double transgenic mice. We found that
aSMA-positive cells were significantly increased in SDF-Tg mice
compared with WT mice as early as the age of 4 months (figure
5A). Similar results were obtained using immunostaining for
aSMA in both SDF-Tg mice and SDF-Tg/IL-1b double transgenic
mice (data not shown). Taken together, these findings suggest
that overexpression of SDF-1 is able to induce the early expansion of aSMA-positive myofibroblasts within the isthmus region
of the gastric corpus.
Given the expansion of stromal cells resulting from the
overexpression of SDF-1 in the stomach, we analysed mRNA
expression of CXCR4 and Gremlin 1 (Grem1). Gremlin 1, a bone
morphogenetic protein antagonist, is a recently identified
marker of MSCs and is highly expressed by cancer-associated
stromal cells.19 35 While SDF-Tg mice alone showed a small but
significant increase in Grem1 mRNA expression, SDF-Tg mice
infected with H felis showed significant upregulation of
both Grem1 and CXCR4 mRNA expression in the stomach
(figure 2C). These findings are consistent with SDF-1-dependent
expansion and recruitment of MSCs to the stomach as recently
described.19 A subset of MSCs has been shown to strongly
express CXCR4 capable of promoting migration to BM.36 We
confirmed Grem1 expression on CXCR4 positive stromal cells, by
Grem1 immunostaining of our CXCR4-EGFP mouse (supplementary figure 9B) and that these Grem1 positive cells were BMderived (figure 5B). We also showed a positive correlation of
mRNA expression of CXCR4 (Spearman rank correlation test,
r¼0.9, p¼0.002). mRNA expression of Grem1 was upregulated
Shibata W, Ariyama H, Westphalen CB, et al. Gut (2012). doi:10.1136/gutjnl-2011-301824
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Figure 4 (A) Proliferation in WT and SDF-Tg mice stomachs stained with Ki67 (n¼5 per group). (B) WT and SDF-Tg mice with or without AMD3100
treatment stained for Ki67. The number of Ki67-positive cells per gland was determined by counting 10 glands per mouse (n¼5 per group, 3-month-old
mice). (C) WT and SDF-Tg mice treated with or without AMD3100 were stained with phospho-Akt or phospho-Erk (n¼5 per group). Data shown are
mean6SE. *p<0.05. Scale bars¼100 mm.
in the stomach of SDF-Tg mice compared with WT mice (figure
5C). We also used immunohistochemistry to confirm the
recruitment of Grem1 (+) MSCs to the gastric mucosa of SDFTg mice in combination with H felis or IL-1b overexpression
(figure 6A). Finally, we examined the direct chemotactic ability
of SDF-1 on BM-MSCs in a migration assay using isolated BMMSCs stimulated with mouse gastric extracts from WT or SDFTg mice in a Boyden chamber assay. Gastric protein extracts
isolated from SDF-Tg mice induced a significant increase in cell
migration of BM-MSCs compared with those isolated from WT
mice, and this migration rate was reduced by the CXCR4
inhibitor AMD3100 (figure 6B). These findings clearly show that
chemotaxis induced by the gastric microenvironment found in
our SDF-Tg mice was mediated, at least in part, through the
SDF1-CXCR4 pathway.
DISCUSSION
While SDF-1/CXCR4 signalling has been linked to numerous
pathophysiological processes, the consequences of upregulated
expression have not been well defined. In the current study we
show that SDF-1 overexpression under the control of the H/KATPaseb promoter does not lead to severe inflammation but,
nevertheless, is able to induce gastric dysplasia and tumour
formation. Overexpression of SDF-1 directly stimulates the
proliferation of gastric epithelial progenitor cells with increases
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in Ki67 and CXCR4-positive cells in the progenitor zone and an
expansion of the K19-positive lineage. In addition, SDF-1 overexpression increased CXCR4-positive fibroblastic cells, especially
aSMA-positive myofibroblasts, probably through recruitment of
Grem1+ MSCs. Despite the limited effect on the induction of
inflammation, SDF-1 overexpression appeared to synergise with
either H felis infection or IL-1b overexpression, resulting in
severe inflammation with a marked increase of F4/80-positive
macrophages. Overexpression of SDF-1 also appeared to synergise with proinflammatory cytokines through the activation of
macrophages and promotion of macrophage survival. Taken
together, our data offer direct evidence that elevation of a single
CXC chemokine, SDF-1, promotes gastric carcinogenesis
through both direct effects on gastric epithelial progenitors and
through modulation of the gastric progenitor niche, consistent
with prior clinical evidence that high levels of SDF-1 in patients
are associated with gastric cancer (figure 7).37
SDF-1 has been thought to be the key chemokine responsible
for the mobilisation and recruitment of inflammatory cells from
the BM to sites of inflammation or cancer.17 SDF-1 is upregulated in a number of inflammatory diseases.18 Deletion of the
CXCR4 receptor in specific haematopoietic subsets clearly
impairs the recruitment of these cells, indicating that the SDF1-CXCR4 axis is required for normal leucocyte trafficking.38 Our
data, however, suggest that high levels of SDF-1 in peripheral
Shibata W, Ariyama H, Westphalen CB, et al. Gut (2012). doi:10.1136/gutjnl-2011-301824
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Gastric cancer
Figure 5 (A) a-smooth muscle actin (aSMA)-positive cells were expanded in SDF-Tg mice. Frozen sections from aSMA/RFP mice crossed with
either WT or SDF-Tg mice were observed under a fluorescent microscope (n¼3 per group). Red: red fluorescent protein (RFP); blue: 49 ,6-diamidino-2phenylindole (DAPI). Original magnification 3100. (B) UBC-GFP traced BM-derived cells in the gastric stroma express Grem1 by immunostaining.
Gastric tissue from SDF-Tg mice transplanted with UBC-GFP bone marrow was stained with anti-Gremlin 1 antibody. Green: bone marrow-derived
cells; red: Gremlin 1. (C) mRNA expression of Gremlin 1 was increased in SDF-Tg mice compared with WT mice at 6 months (n¼4 per group). Results
shown are mean+SE. *p<0.05. Scale bars¼100 mm.
tissues such as the stomach are not sufficient for recruitment of
leucocytes, since the SDF-1 Tg mice showed little inflammation
at early time points. It was only at later time points that we
observed a significant increase in F4/80-positive macrophages,
and SDF-1 overexpression in combination of IL-1b overexpression resulted in a much more severe inflammatory
response. It is likely that part of the explanation relates to the
endogenous levels of BM SDF-1 which prevented the egress of
BM cells.39 The key to the recruitment of inflammatory cells
from BM may depend as much on the degradation of BM SDF-1
in response to proinflammatory cytokines such as IL-1b as it
does on high levels of SDF-1 in peripheral tissue.25 40 In addition,
our data would suggest that SDF-1 might contribute to
inflammation by activating and stabilising macrophages, along
with its effects on chemotaxis, consistent with previous
reports.41 42
Our data support the notion that one target of SDF-1
signalling is directly on gastric epithelial progenitors. We found
CXCR4-positive cells in the isthmus of the normal gastric
corpus and in the lower third of the normal antral glands. These
portions of the gastric corpus and antrum contain gastric stem
and progenitor cells, and also the Ki67-positive progenitor cells.
In addition, in SDF-1 Tg mice we observed a gradual increase in
CXCR4-EGFP-positive epithelial cells, consistent with an
amplification of CXCR4-positive progenitors. Thus, our findings
suggest that, in the gastric epithelium, SDF-1 secreted by gastric
myofibroblasts regulates the proliferation and possibly the
location of gastric epithelial progenitors. CXCR4 expression has
been observed in the CNS and has been postulated to be
important in regulating the migration of progenitor cells in
postnatal brain.32 The downward migration of the proliferative
zone commonly observed during gastric preneoplasia, for
example, could in theory be related in part to SDF-1 expression
by MSC-associated stromal cells which tend to expand at the
base of the gastric glands. CXCR4 is also found in many cancer
cell lines where it has been associated with tumour growth and
metastasis,43 and the expression of CXCR4 by gastric progenitors could account for this finding of expression in cancer cells.
However, the most prominent effects of SDF-1 appeared to be
the expansion of aSMA-positive myofibroblasts in the gastric
mucosa. At early time points, much of this expansion appears to
be due to proliferation of resident tissue mesenchymal cells,
since we found only a small number of BM-derived aSMApositive myofibroblasts in our BM transplantation studies.
Nevertheless, the data are consistent with the likely presence of
MSCs in most peripheral tissues such as the stomach and the
slow time course for BM-derived cell recruitment prior to the
development of dysplasia.19 In previous studies we established
Gremlin 1 as a putative marker for MSCs that give rise to
myofibroblasts.19 Indeed, in the current study we found that
Grem1 mRNA expression was significantly upregulated in SDFTg mice when compared with WT mice, particularly in the
setting of H felis. We also showed that Grem1+ MSCs express
CXCR4 and migrate in response to SDF-1 expression. In
previous studies we also demonstrated that CXCR4 function
was integral to the development of myofibroblasts as well, since
TGFb can induce myofibroblastic differentiation from MSCs
through upregulation of SDF-1 production and is inhibited by
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Gastric cancer
Figure 6 (A) Gremlin 1-positive cells were increased in SDF-Tg mice compared with WT mice infected with Helicobacter felis. Representative
paraffin sections from stomachs are shown (top). Gremlin 1-positive cells were increased in SDF/H/K-ATPase-IL-1b double transgenic mice compared
with H/K-ATPase-IL-1b mice (bottom). Frozen sections from SDF/H/K-ATPase-IL-1b or H/K-ATPase-IL-1b mice were stained (n¼3 per group). Green:
Gremlin 1; blue: 49 ,6-diamidino-2-phenylindole (DAPI). Original magnification 3100. (B) Transwell migration assay demonstrated the migration of bone
marrow mesenchymal stem cells (BM-MSCs) in response to stromal cell-derived factor-1 (SDF-1) in 3-month-old mice. BM-MSCs were incubated with
the gastric mixture from WT or SDF-Tg mice. SDF-1-induced BM-MSC migration was significantly inhibited by AMD3100. Results shown are mean
+SE. *p<0.05 in each comparison indicated.
the CXCR4 antagonist AMD3100.19 While CXCR4 antagonism
could also inhibit non-SDF-1 ligands such as ubiquitin, we
believe that, in our SDF1-Tg driven model, the consequences of
CXCR4 antagonism are primarily due to the loss of SDF1-related
signalling.
In conclusion, the results presented here show that SDF-1/
CXCR4 signalling is important to the gastric epithelial niche.
SDF-1 is normally produced by MSC-associated stromal
cells, and increased SDF-1 promotes the proliferation of
both epithelial cells and the expansion of stromal cells. While
SDF-1 is a weak inflammatory chemokine, upregulation of
SDF-1/CXCR4 pathway can synergise with other proinflammatory molecules (such as IL-1b) and thus contribute to
neoplasia. Taken together, the SDF/CXCR4 signalling pathway
represents a promising target for future cancer prevention and
treatment.
Figure 7 Schematic hypothesis of
stromal cell-derived factor-1 (SDF-1)induced carcinogenesis. IL, interleukin;
MSCs, mesenchymal stem cells;
aSMA, a-smooth muscle actin.
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Gastric cancer
Acknowledgements The authors thank Kelly S Betz, Ashley Whelan, Justin
DeGrazia and Chintan Kapadia for their help with animal studies.
19.
Contributors Study concept and design, obtained funding and study supervision:
TCW. Acquisition of data: WS, HA. Analysis and interpretation of data: WS, HA, CBW,
DW, JF, SM, SA, ZD, MQ, TCW. Drafting of manuscript: WS, HA, CBW, DW, MQ,
TCW.
20.
Funding This research was supported by grants from the National Institute of Health
grants 5R01CA093405-08 (TCW). WS was supported by the Japan Society for the
Promotion of Science. MQ is supported by the Deutsche Krebshilfe.
Competing interests None.
21.
22.
23.
24.
Provenance and peer review Not commissioned; externally peer reviewed.
25.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Fox JG, Wang TC. Inflammation, atrophy, and gastric cancer. J Clin Invest
2007;117:60e9.
Nickoloff BJ, Ben-Neriah Y, Pikarsky E. Inflammation and cancer: is the link as
simple as we think? J Invest Dermatol 2005;124:xexiv.
Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell
2010;140:883e99.
Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and
bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature
1996;382:635e8.
Lazarini F, Tham TN, Casanova P, et al. Role of the alpha-chemokine stromal cellderived factor (SDF-1) in the developing and mature central nervous system. Glia
2003;42:139e48.
Zou YR, Kottmann AH, Kuroda M, et al. Function of the chemokine receptor CXCR4
in haematopoiesis and in cerebellar development. Nature 1998;393:595e9.
Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential
for vascularization of the gastrointestinal tract. Nature 1998;393:591e4.
De Clercq E. The AMD3100 story: the path to the discovery of a stem cell mobilizer
(Mozobil). Biochem Pharmacol 2009;77:1655e64.
Eash KJ, Means JM, White DW, et al. CXCR4 is a key regulator of neutrophil release
from the bone marrow under basal and stress granulopoiesis conditions. Blood
2009;113:4711e19.
Petit I, Jin D, Rafii S. The SDF-1-CXCR4 signaling pathway: a molecular hub
modulating neo-angiogenesis. Trends Immunol 2007;28:299e307.
Duda DG, Kozin SV, Kirkpatrick ND, et al. CXCL12 (SDF1alpha)-CXCR4/CXCR7
pathway inhibition: an emerging sensitizer for anticancer therapies? Clin Cancer Res
2011;17:2074e80.
Hattori K, Heissig B, Tashiro K, et al. Plasma elevation of stromal cell-derived
factor-1 induces mobilization of mature and immature hematopoietic progenitor
and stem cells. Blood 2001;97:3354e60.
Hiasa K, Ishibashi M, Ohtani K, et al. Gene transfer of stromal cell-derived
factor-1alpha enhances ischemic vasculogenesis and angiogenesis via vascular
endothelial growth factor/endothelial nitric oxide synthase-related pathway:
next-generation chemokine therapy for therapeutic neovascularization. Circulation
2004;109:2454e61.
Yamaguchi J, Kusano KF, Masuo O, et al. Stromal cell-derived factor-1 effects on
ex vivo expanded endothelial progenitor cell recruitment for ischemic
neovascularization. Circulation 2003;107:1322e8.
Karin N. The multiple faces of CXCL12 (SDF-1alpha) in the regulation of immunity
during health and disease. J Leukoc Biol 2010;88:463e73.
Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540e50.
Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive
human breast carcinomas promote tumor growth and angiogenesis through
elevated SDF-1/CXCL12 secretion. Cell 2005;121:335e48.
Tu S, Bhagat G, Cui G, et al. Overexpression of interleukin-1beta induces gastric
inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice.
Cancer Cell 2008;14:408e19.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Quante M, Tu SP, Tomita H, et al. Bone marrow-derived myofibroblasts contribute to
the mesenchymal stem cell niche and promote tumor growth. Cancer Cell
2011;19:257e72.
Balkwill F. The significance of cancer cell expression of the chemokine receptor
CXCR4. Semin Cancer Biol 2004;14:171e9.
Mishra PJ, Humeniuk R, Medina DJ, et al. Carcinoma-associated fibroblast-like
differentiation of human mesenchymal stem cells. Cancer Res 2008;68:4331e9.
Spaeth EL, Dembinski JL, Sasser AK, et al. Mesenchymal stem cell transition to
tumor-associated fibroblasts contributes to fibrovascular network expansion and
tumor progression. PloS One 2009;4:e4992.
Direkze NC, Hodivala-Dilke K, Jeffery R, et al. Bone marrow contribution to tumorassociated myofibroblasts and fibroblasts. Cancer Res 2004;64:8492e5.
Mishra P, Banerjee D, Ben-Baruch A. Chemokines at the crossroads of tumorfibroblast interactions that promote malignancy. J Leukoc Biol 2011;89:31e9.
Kerfoot SM, Andonegui G, Bonder CS, et al. Exogenous stromal cell-derived factor-1
induces modest leukocyte recruitment in vivo. Am J Physiol Heart Circ Physiol
2008;294:H2524e34.
Rogers AB, Houghton J. Helicobacter-based mouse models of digestive system
carcinogenesis. Methods Mol Biol 2009;511:267e95.
El-Omar EM, Carrington M, Chow WH, et al. Interleukin-1 polymorphisms
associated with increased risk of gastric cancer. Nature 2000;404:398e402.
Brembeck FH, Moffett J, Wang TC, et al. The keratin 19 promoter is potent for cellspecific targeting of genes in transgenic mice. Gastroenterology 2001;120:1720e8.
Quante M, Marrache F, Goldenring JR, et al. TFF2 mRNA transcript expression
marks a gland progenitor cell of the gastric oxyntic mucosa. Gastroenterology
2010;139:2018e27.e2.
Lu DY, Tang CH, Yeh WL, et al. SDF-1alpha up-regulates interleukin-6 through
CXCR4, PI3K/Akt, ERK, and NF-kappaB-dependent pathway in microglia. Eur J
Pharmacol 2009;613:146e54.
Dubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, et al. Secreted trefoil factor 2
activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol
Chem 2009;284:3650e62.
Tran PB, Banisadr G, Ren D, et al. Chemokine receptor expression by neural
progenitor cells in neurogenic regions of mouse brain. J Comp Neurol
2007;500:1007e33.
Barker N, Huch M, Kujala P, et al. Lgr5(+ve) stem cells drive self-renewal in the
stomach and build long-lived gastric units in vitro. Cell Stem Cell 2010;6:25e36.
Brittan M, Wright NA. Gastrointestinal stem cells. J Pathol 2002;197:492e509.
Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogenetic protein
antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can
promote tumor cell proliferation. Proc Natl Acad Sci U S A 2006;103:14842e7.
Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem
cells strongly expresses functionally active CXCR4 receptor capable of promoting
migration to bone marrow. Blood 2004;104:2643e5.
Iwasa S, Yanagawa T, Fan J, et al. Expression of CXCR4 and its ligand SDF-1 in
intestinal-type gastric cancer is associated with lymph node and liver metastasis.
Anticancer Res 2009;29:4751e8.
Sugiyama T, Kohara H, Noda M, et al. Maintenance of the hematopoietic stem cell
pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches.
Immunity 2006;25:977e88.
Cashen AF, Lazarus HM, Devine SM. Mobilizing stem cells from normal donors: is it
possible to improve upon G-CSF? Bone Marrow Transplant 2007;39:577e88.
Broxmeyer HE, Orschell CM, Clapp DW, et al. Rapid mobilization of murine and
human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist.
J Exp Med 2005;201:1307e18.
Lee Y, Gotoh A, Kwon HJ, et al. Enhancement of intracellular signaling associated
with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy
with other cytokines. Blood 2002;99:4307e17.
Hodohara K, Fujii N, Yamamoto N, et al. Stromal cell-derived factor-1 (SDF-1) acts
together with thrombopoietin to enhance the development of megakaryocytic
progenitor cells (CFU-MK). Blood 2000;95:769e75.
Yasumoto K, Koizumi K, Kawashima A, et al. Role of the CXCL12/CXCR4 axis in
peritoneal carcinomatosis of gastric cancer. Cancer Res 2006;66:2181e7.
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Stromal cell-derived factor-1 overexpression
induces gastric dysplasia through
expansion of stromal myofibroblasts and
epithelial progenitors
Wataru Shibata, Hiroshi Ariyama, Christoph Benedikt Westphalen, et al.
Gut published online February 23, 2012
doi: 10.1136/gutjnl-2011-301824
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